She Said I Think I'll Go To Boston

T-minus two weeks, and counting. I wish I could have a moment of certainty and make a decision about the college I'll go to. But I suppose that would be too much to ask for. Instead, next week I'm going to attend an admitted students day at Tufts, so hopefully I'll closer to my decision soon. (But, let's be honest, May 1st is getting closer and closer whether I like it or not.)

Although it's been nice sleeping in, I miss going into the lab and doing something with my time. (Maybe I'll have to get a job soon so I have something to do. Yay...) All in all, I've had too much free time to sit around and think about the future. This includes the near future as well - it turns out I'm in the first SRP presenting group. I'll talk about my project and my experience on May 9th any time between 10am and 11am. At least I have something else to worry about now, besides college.

On a different note, my uncle took Dora to the vet this week. Since we're not the best people to take care of a wild dove, we gave her to the vet, who promised that she'd find a rehabilitation place for for her. It's nice to know that Dora will be returned to the wild, but she'll be missed all the same (except, probably, by my aunt).

I'm going to save my results for my final blog post (next week's post) and for my SRP presentation, so stay tuned. What will I talk about in this post then, you might ask. I asked myself this too, and after much discussion with myself (I'm not crazy; the voices in my head told me so) we decided to explain the graphs more. Before you get too excited (and yes, I can sense your enthusiasm), you should know that this means I'm guaranteed to add pictures along with the words, instead of just blocks of text.

That moment when it's easier to edit a picture in PowerPoint than Paint....

The pH on the side of the graph isn't exactly correct; the conversation from the voltage in the pH electrodes to the pH needs to be altered. The actual pH is around 6 to 7. We haven't fixed the conversion because we're looking for the change in pH, not the pH itself.

The first time I saw a graph like this, I was extremely confused. (And I didn't even have the arrows to help me.) I mean, sure, it looks cool - but it was just a bunch of peaks and valleys to me. If you're anything like me, you'd be wondering what, exactly, is this means in English. (And if you're not, I'm afraid you still have to put up with my questions and answers.)

Valinomycin is a potassium ion-selective transporter; it only moves potassium ions. It looks a little like this:

It looks so simple.... I've yet to figure out how it actually works.

The first graph I showed you is a blank, meaning it has no M2 protein channel, and it also has no drug added. The liposomes (hopefully) have more potassium ions than the external solution, while the external solution (in theory) has more protons than the liposomes. This sets up a gradient; the potassium ions want to leave the liposomes, and the protons want to enter the liposomes. However, without a channel, the ions can't move.

This is where valinomycin comes in. (Literally.) It's first added to depolarize the liposomes (meaning that protons enter the liposomes). It allows the ions to move - but only if there is a M2 channel. Therefore, in the blanks, we expect there to be no valinomycin signal (and by "signal," I mean change in pH). And, if you look back to the graph, you'll see that this is true. You could argue that there is, in fact, a small change in pH, but I'd tell you to look at the second addition of valinomycin. Both changes in pH are the same. We can assume that the pH change is from something else, instead of the uptake of protons into the liposomes. And it is; the valinomycin is in an ethanol solvent, which is basic. Adding ethanol to this experiment would produce a similar pH change.

Conversely, we would expect a valinomycin signal in a liposome with M2 channels.

Yay, the valinomycin signal is different. We're done now, right?

I'm afraid that there's more to the graphs than the valinomycin signal. (There're three different colored arrows; you should have expected this.) If you compare the graphs, you'll notice that not only did the valinomycin signal increase, the CCCP signal decreased.

CCCP is a proton-selective transporter, and it acts like a M2 channel. It allows protons to come into the liposomes, resulting in a small change in the pH of the external solution. In the blanks, we expect a CCCP signal, because the liposomes are not depolarized by the valinomycin. The CCCP depolarizes the liposomes in the blanks, instead of the valinomycin. In the liposomes with M2 channels, there is no CCCP signal, because the liposomes are already depolarized by valinomycin. (Again, the CCCP "signal" is due to the ethanol, instead of proton flux.)

The previous graph has liposomes with M2 channels, and the next graph has liposomes with M2 channels and a drug. Kudos to the person who can guess what kind of "signals" we're looking for. (Hint: A good drug will block proton flux.) (Another hint: CCCP will depolarize the liposomes, whether or not there are M2 channels present.)

Am I the only one who would look at the graphs and this painstakingly detailed explanation, and ask what the HCl is for?

If you guessed that a good drug would reduce the valinomycin signal, you win! (If you didn't, at least you tried, right?) My whole project was based on the comparison of these graphs. I put my data into an Excel spreadsheet, and somehow they got an estimate for proton flux during the addition of valinomycin. Unfortunately, the numbers were pretty subjective, since we changed the numbers for the graphs ourselves by moving the red lines near the valinomycin signal. Fortunately, someone in the lab is working on a program that will automatically take the differences in the pH before and after the addition of valinomycin into account, so the numbers will be less subjective. Naturally, this program will only be used after I've completed my project. (I suppose that's the way the cookie crumbles.)

 

Thank you to anyone who even attempted to read this! I appreciate your efforts. (And if you just skipped to the end, and saw the word "cookie" - well, no cookies for you, then.) Feel free to ask me any questions.

Until next time!

- Lauren